PROJECT SUMMARY/ABSTRACT Immune checkpoint inhibition (ICI) has improved the outlook for many patients with metastatic cancer. However, resistance to ICI remains poorly understood, with no currently approved therapeutic strategies to overcome resistance. A possible mechanism of resistance to ICI is ineffective recognition of a tumor by a T cell, which is mediated by Major Histocompatibility Complex (MHC)-T cell receptor interactions. Most tumors express MHC class I, which presents antigen to CD8+ T cells, which are thought to be the main cytotoxic effector cells. Interestingly, a subset of tumors also express MHC class II, which presents antigen to CD4+ T cells and is canonically thought to only be present on ?professional? antigen presenting cells of the immune system. CD4+ T cells are a diverse population and include helper subsets that are crucial generating and maintaining an effective immune response. Our lab has shown that tumor-specific MHC-II (tsMHC-II) expression is a positive prognostic indicator for melanoma patients treated with ICI. Additionally, our preliminary data show that enforced tumor cell expression of Class II Major Histocompatibility Complex Transactivator (CIITA), which drives expression of MHC- II and related machinery, enhances tumor rejection in immunocompetent mice. Additional preliminary co-culture experiments suggest that CD4+ T cells produce more pro-inflammatory cytokine when stimulated with MHC-II+ compared to MHC-II- tumor cells. These data, in mouse and human, suggest that tsMHC-II may enhance anti- tumor immunity through activation of helper T cells. Thus, we hypothesize that tsMHC-II can be recognized by T cell receptors (TCRs) from CD4+ T cells, activates signaling in CD4+ T cells, and skews toward anti-tumor polarization of CD4+ T cells. This proposal outlines two detailed specific aims for testing this hypothesis. Proposed experiments include in vitro co-culture of tumor cells and T cells in order to isolate the phenotypic change in CD4+T cells following engagement of tsMHC-II. In vivo murine models and mass cytometry-based characterization of human tumors will further define how tsMHC-II alters the immune microenvironment. Furthermore, we will generate solubilized TCRs (sTCRs) from CD4+ T cells infiltrating tsMHC-II+ tumors and test the ability of these sTCRs to bind to autologous tsMHC-II+ tumor cells. We will also transduce these TCRs into an immortalized T cell line to test the ability of tsMHC-II engagement to induce signaling changes in T cells. Completion of this project will yield new insights regarding how tsMHC-II is recognized by autologous infiltrating CD4+ T cells and how tsMHC-II influences anti-tumor CD4+ T cell responses. Overall, this project will enhance understanding of anti-tumor immunity and may suggest novel translational approaches to circumventing resistance to ICI in the clinic through tsMHC-II upregulating agents.